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Isoflurane and Halothane Attenuate Endothelium-Dependent Vasodilation in Rat Coronary Microvessels

Park, Kyung W. MD; Dai, Hai B. MD; Lowenstein, Edward MD; Darvish, Amir MS; Sellke, Frank W. MD

Cardiovascular Anesthesia
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Volatile anesthetics attenuate endothelium-dependent vasodilation but the mechanism of attenuation remains controversial. The present study examines the mechanism of isoflurane- and halothane-mediated attenuation of endothelium-dependent vasodilation in Wistar rat coronary microvessels of about 100 micro m internal diameter. The vessels were studied in vitro in a pressurized (40 mm Hg), no-flow state using video microscopy. After preconstriction of the vessels with the thromboxane analog U46619 1 micro M, concentration response curves to acetylcholine (ACh), the calcium ionophore A23187, sodium nitroprusside (SNP), or the stable cyclic guanosine monophosphate (cGMP) analog 8-bromo-cGMP (Br-cGMP) were obtained in the presence of 0% (control), 1% or 2% isoflurane, or 1% or 2% halothane. Isoflurane 1% and 2% significantly attenuated vasodilation to ACh and A23187. Isoflurane 2%, but not 1%, attenuated vasodilation to SNP. Vasodilation to Br-cGMP was not affected by isoflurane. Halothane attenuated vasodilation to ACh, but had no effect on vasodilation to A23187, SNP, or Br-cGMP. We conclude that isoflurane attenuates endothelium-dependent vasodilation by impairing at least two distinct steps in the nitric oxide (NO)-cGMP pathway, the first being between endothelial increase of calcium and smooth muscle guanylate cyclase and the second being inhibition of soluble guanylate cyclase activity. These two steps appear to have different sensitivities to the effect of isoflurane. Halothane has an effect at the endothelial receptor level, but not any distal steps in the NO-cGMP pathway.

(Anesth Analg 1997;84:278-84)

Departments of (Park, Lowenstein, Darvish) Anesthesia and Critical Care and (Dai, Sellke) Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.

Supported in part by United States Public Health Service Grant HL-46716 and by a grant from Beth Israel Deaconess Anesthesia Foundation.

Accepted for publication September 25, 1996.

Address correspondence and reprint requests to Kyung W. Park, MD, Department of Anesthesia & Critical Care, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA 02215.

Since Muldoon et al. [1] demonstrated that halothane attenuated endothelium-dependent relaxation by ACh, others have reported a similar finding with halothane and isoflurane [2,3]. However, there are conflicting reports on how the volatile anesthetics attenuate endothelium-dependent relaxation. Thus Toda et al. [2] reported that halothane and isoflurane do not attenuate receptor-independent, endothelium-dependent relaxation of isolated rat aortic rings by the calcium ionophore A23187, whereas Uggeri et al. [3] reported a significant effect of the anesthetics on A23187-induced relaxation in similar preparations. Furthermore, while both these authors reported a lack of attenuation of sodium nitroprusside (SNP)-mediated, endothelium-independent relaxation by isoflurane or halothane, others [4,5] demonstrated, in rat aortic rings, that both halothane and isoflurane interfere with stimulation of smooth muscle guanylate cyclase by nitric oxide (NO). In addition, Blaise and To [6] suggested that isoflurane interferes with NO-mediated relaxation at a site distal to the endothelium, either by diminishing the stability of NO or its action on vascular smooth muscle (VSM), presumably via VSM guanylate cyclase.

Although all these studies used preparations of conductance arteries, small arteries and arterioles that constitute resistance arteries primarily determine vascular resistance and regulate flow distribution in vascular beds. In the present study, we examined the effects of the volatile anesthetics, isoflurane and halothane, on endothelium-dependent vasodilation in resistance coronary arteries and sought to define the mechanism(s) of any observed attenuation. Our hypotheses were that the volatile anesthetics attenuate endothelium-dependent dilation in coronary resistance arteries and that the mechanism of attenuation may involve multiple steps in the NO-cyclic guanosine monophosphate (cGMP) pathway, distal to agonist activation of the endothelial receptor.

To test our hypotheses, we monitored changes in diameter of resistance coronary arteries of about 100 micro m in diameter, in a no-flow preparation using a video detection system. Various advantages of this set-up in comparison to measurement of isometric tension have been discussed previously [7]; however, one advantage particularly pertinent to this study is that in a no-flow preparation we remove any confounding influences of flow-mediated release of NO [8]. We first examined whether there is a significant contribution of prostanoid(s) in endothelium-dependent dilation in our vessel preparation. After demonstrating negligible contribution of prostanoids, we then examined four vasodilators that act in the NO-cGMP pathway, either in the presence or absence of isoflurane or halothane 1% or 2%. Acetylcholine stimulates the endothelial muscarinic receptor to increase endothelial Ca2+ concentration, which in turn stimulates endothelial NO synthase [9]. The calcium ionophore A23187 increases endothelial Ca2+ concentration via a non-receptor-mediated mechanism and thus stimulates NO synthase [2]. Endothelium-derived NO diffuses into the VSM, where it activates guanylate cyclase, producing cGMP from guanosine triphosphate. SNP is an endothelium-independent NO donor [10]. Br-cGMP is a nonhydrolyzable analog of cGMP, and like cGMP, activates cGMP-dependent protein kinase [11]. Activation of cGMP-dependent protein kinase brings about vasodilation [12]. Differential effects of anesthetics on vasodilation produced by these dilators would point to the site(s) of anesthetic action in the NO-cGMP pathway.

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Methods

Vessel Preparation

In accordance with institutional animal care committee standards, Wistar rats of either sex, weighing 100-150 g, were anesthetized by injecting ketamine 40 mg/kg and xylazine 5 mg/kg intraperitoneally. Their hearts were quickly harvested and placed in cold (4 degrees C) modified Krebs buffer (NaCl 120 mM, KCl 5.9 mM, dextrose 11.1 mM, NaHCO3 25 mM, NaH2 PO4 1.2 mM, MgSO4 1.2 mM, CaCl2 2.5 mM). Subepicardial microvessels that were third or fourth generation branches of the left anterior descending artery were dissected carefully from the surrounding myocardial tissue. Each vessel was placed in a vessel chamber, cannulated with dual micropipettes measuring 50-75 micro m in diameter, and secured with a 10-0 Ethilon[TM] (Ethicon Inc., Somerville, NJ) suture. The vessel was continuously bathed with modified Krebs buffer, gassed with 95% O2/5% CO2 mixture, and maintained at 36.5-37.5 degrees C and pH of 7.35-7.45. PO2 in the vessel chamber exceeded 400 mm Hg. As the vessel was studied in a no-flow state, the pressure in the micropipettes was maintained at 40 mm Hg to provide distention. The vessel was visualized and its internal lumen diameter was measured and recorded, as previously described [13]. Stability of similarly prepared vessel preparations over at least 2.5 h has been demonstrated previously [7].

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Contribution of NO to Endothelium-Dependent Vasodilation of Rat Coronary Microvessels

To determine the relative importance of nitric oxide and prostacyclin in endothelium-dependent vasodilation of our vessel preparation, 19 vessels were studied. Each vessel was equilibrated in the vessel chamber for a minimum of 20-30 min, either in the presence of the cyclooxygenase inhibitor indomethacin 10 micro M (n = 6, baseline diameter 100 +/- 10 micro m), the NO synthase inhibitor NG-nitro-L-arginine (L-NNA) 10 micro M (n = 6, baseline diameter 107 +/- 10 micro m), or neither (n = 7, baseline diameter 102 +/- 9 micro m). A baseline diameter (Dbaseline) was measured at the end of initial equilibration. Each vessel was then preconstricted with the thromboxane analog U46619 1 micro M and the constricted diameter (Dconst) was measured. In our previous studies [7,13,14], U46619 was shown to produce consistent, reproducible constriction of coronary microvessels. Each vessel was then subjected to increasing concentrations of acetylcholine (ACh) 10-8-10-4 mol/L for 2 min at each concentration. At each concentration, the internal diameter was measured (Drelax) and percent relaxation from U46619-induced preconstriction was calculated: Equation 1 At the end of each experiment, the vessel chamber was flushed with fresh Krebs buffer and the vessel requilibrated at 37 degrees C. KCl was then added to a final concentration of 100 mM and the internal lumen diameter was measured. Only those vessels that constricted by at least 15% to KCl at the end of each experiment were considered still viable and included for data analysis.

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Effect of Anesthetics

After microvessels were equilibrated for a minimum of 20-30 min in the vessel chamber, a baseline measurement of the vessel lumen internal diameter (Dbaseline) was obtained. The vessel was then preconstricted with the thromboxane analog U46619 1 micro M for 5 min. Only those vessels that constricted by 20%-40% to U46619 were studied further. The vessel was then subjected to either isoflurane 1% or 2%, halothane 1% or 2%, (study groups), or neither (control), by adding the anesthetic to the gas mixture bubbling the Krebs buffer solution, using an in-line bubble-through vaporizer.

In a preliminary experiment, using gas chromatography, it took less than 10 and 15 min for isoflurane and halothane, respectively, to reach a steady-state concentration after it was introduced in the vessel chamber [7]. The anesthetic content in the gas mixture was continuously monitored using a Rascal II[R] Gas Analyzer (Ohmeda, Salt Lake City, UT), previously calibrated with industrial standards. Previously we demonstrated by gas chromatography analysis that in our experimental preparation, the millimolar concentration and partial pressure of isoflurane or halothane in the vessel chamber reflected its concentration in the gas mixture bubbled into the buffer solution [7].

No significant change in internal diameter of the U46619-preconstricted vessel was noted after steady state concentrations of isoflurane 1% or 2% were obtained. In contrast, there was 5%-7% relaxation of the U46619-preconstricted vessel after steady-state concentrations of halothane 1% or 2% were obtained. The diameter obtained after U46619 and either isoflurane or halothane (or neither anesthetic in case of a control vessel) was considered as the constricted diameter (Dconst).

At least 15 min after introduction of isoflurane or 20 min after introduction of halothane, the vessel was subjected to increasing concentrations of ACh 10-8-10-4 mol/L, the calcium ionophore A23187 10-8-10-5 mol/L, SNP 10 (-9-10)-6 mol/L, or the stable cGMP analog 8-bromo-cGMP (Br-cGMP) 10-9-10 (-5) mol/L for 2 min at each concentration. Vessel internal diameters were monitored and percent relaxation was calculated as described above. After exposure of a vessel to a dilator, the vessel was flushed with fresh Krebs buffer and the diameter returned to baseline. Viability of the vessels was then tested as described above. Each experiment took less than 2 h.

Additionally, we examined whether isoflurane or halothane-mediated attenuation of endothelium-dependent vasodilation is reversible after the anesthetic is turned off. Coronary microvessels were dissected and equilibrated in a vessel chamber as described above. Then, the vessel was subjected to isoflurane 2%, halothane 2%, or neither (control) in 95% O2/5% CO2 for 30 min. The anesthetic was turned off and the vessel was flushed with fresh Krebs buffer and reequilibrated at 37 degrees C. A baseline diameter (Dbaseline) was measured. The vessel was preconstricted with U46619 1 micro M and the constricted diameter (Dconst) was measured. Then the vessel was subjected to ACh 10-7 M or 10-5 M, A23187 10-7.5 M or 10-6 M, or SNP 10-8 M or 10-6 M. Vessel internal diameters were monitored and percent relaxation was calculated as described above. Viability of the vessels was tested as described above. Thirty-nine vessels (103 +/- 12 micro m) from 16 rats were the subject of this portion of our study.

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Statistical Analysis

No animal contributed more than one vessel to any one experimental group. Therefore, n for each group represents the number of animals as well as the number of vessels. All data are presented as mean +/- SD.

Whether there is a concentration-dependent relaxation to a vasodilator was tested by one-way analysis of variance (ANOVA) (linear contrast). The effects of isoflurane or halothane on concentration response curves to the various vasodilators tested were analyzed by two-way ANOVA (blocked design), with post-hoc multiple pairwise comparison (Newman-Keuls) and stratified z tests to identify the concentrations where the differences in response were significant. The null hypothesis tested was that isoflurane or halothane had no effect on the response of the vessels to the vasodilators. Similarly, the effects of the inhibitors L-NNA or indomethacin on concentration response curves were analyzed by two-way ANOVA (blocked design). Where appropriate, two-tailed Student's t-test was used to compare the means of two groups. Significance was considered as P < 0.05. All statistics were calculated using True Epistat[TM] software (Epistat Services, Richardson, TX).

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Results

Preconstricted rat subepicardial arteries demonstrated a concentration-dependent dilation to ACh. This effect was not significantly altered by the cyclooxygenase inhibitor indomethacin (Figure 1). The ACh-induced dilation was abolished by the NO synthase inhibitor L-NNA (P < 0.01) and was converted to a contractile response (P < 0.05) (control group: n = 7, baseline size 102 +/- 9 micro m [mean +/- SD]; indomethacin-treated group: n = 6, size 100 +/- 10 micro m; L-NNA-treated group: n = 6, size 107 +/- 10 micro m).

Figure 1

Figure 1

Vasodilation of rat subepicardial arteries by the endothelium-dependent receptor-mediated vasodilator ACh (control group: n = 7, baseline size 102 +/- 9 micro m) was attenuated by either isoflurane 1% (n = 8, size 100 +/- 7 micro m) or 2% (n = 6, size 102 +/- 13 micro m) (Figure 2A). Vasodilation to ACh was attenuated by halothane 2% (n = 5, size 99 +/- 14 micro m), but not 1% (n = 5, size 87 +/- 11 micro m) (Figure 3A). Vasodilation to the endothelium-dependent, nonreceptor-mediated calcium ionophore A23187 (control group: n = 7, size 105 +/- 8 micro m) was attenuated by either isoflurane 1% (n = 7, size 85 +/- 13 micro m) or 2% (n = 6, size 108 +/- 9 micro m) (Figure 2B). Attenuation was greater by isoflurane 2% than by isoflurane 1% (P < 0.05). In contrast, the effect of halothane 1% (n = 6, size 90 +/- 18 micro m) or 2% (n = 6, size 97 +/- 10 micro m) on A23187-mediated vasodilation was not significant (Figure 3B). Vasodilation to the endothelium-independent vasodilator SNP (control group: n = 11, size 105 +/- 5 micro m) was attenuated by isoflurane 2% (n = 5, size 103 +/- 9 micro m), but not isoflurane 1% (n = 7, size 102 +/- 6 micro m) (Figure 2C). In contrast, the effect of halothane 1% (n = 6, size 102 +/- 9 micro m) or 2% (n = 8, size 104 +/- 5 micro m) on SNP-mediated dilation was not significant (Figure 3C).

Figure 2

Figure 2

Figure 3

Figure 3

Vasodilation to the cGMP analog Br-cGMP was not attenuated by either concentration of isoflurane (Figure 2D) or halothane (Figure 3D) (control: n = 6, size 98 +/- 11 micro m; isoflurane 1%: n = 5, size 96 +/- 13 micro m; isoflurane 2%: n = 5, size 105 +/- 12 micro m; halothane 1%: n = 5, size 97 +/- 14 micro m; halothane 2%: n = 6, size 104 +/- 8 micro m).

Prior exposure of the vessels to isoflurane 2% or halothane 2% did not alter their response to ACh, A23187, or SNP (Table 1).

Table 1

Table 1

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Discussion

An extensive discussion on the relative merits of our video detection system in comparison to measurement of isometric tension, such as was used in many previous studies of anesthetic attenuation of endothelium-dependent vasodilation [2-6,15-17], has been previously published [7]. A particularly attractive feature of our preparation in relation to the present study is that the vessels are examined in a no-flow setup. Flow or shear stress on the endothelium releases NO [8] and would have been a confounding influence on endothelium-dependent vasodilation that was eliminated in our preparation.

Furthermore, unlike in previous studies of anesthetic attenuation of endothelium-dependent vasodilation which used conductance vessels [2-5,16,17] or cell cultures derived from conductance vessels [6,15], we used resistance arteries, which primarily regulate vascular resistance and determine flow distribution. Conductance and resistance arteries often have different vasomotor responses to nonanesthetic vasoactive drugs [18,19] and to the volatile anesthetics [7,13]. Halothane and isoflurane, which have similar direct vasomotor effects on conductance coronary arteries, have opposite effects on resistance vessels [7]. It is interesting to note that these two anesthetics, which are reported to have similar attenuating effects on endothelium-dependent vasodilation in conductance vessels [2,3,5,15-17], were found to have distinct mechanisms of attenuation of endothelium-dependent vasodilation in resistance coronary arteries in our study.

The major findings of this study are that isoflurane interferes with at least two distinct steps in the NO-cGMP pathway-the first one at or distal to endothelial increase in Ca2+ and proximal to VSM guanylate cyclase, and the second probably involving guanylate cyclase itself-and that these two steps appear to have different sensitivities to isoflurane with the former being more sensitive than the latter. Our findings do not exclude the possibility that isoflurane may act on more than two steps in the NO-cGMP pathway. Halothane, on the other hand, appears to have an effect at the endothelial receptor level. No distal effect of halothane could be demonstrated in our model. The effect of either anesthetic was reversible once the anesthetic was discontinued.

In our preparation of coronary microvessels, isoflurane 1% or 2% attenuated ACh- or A23187-mediated vasodilation. This finding is consistent with that of Uggeri et al. [3] obtained in larger vessels. In contrast, Toda et al. [2] observed in rat aortic preparations that isoflurane attenuated relaxation due to ACh, but not A23187 (10-7.5-10-6 M). Even in our preparation where a wider concentration range of A23187 (10-8-10-5 M) was examined, the attenuating effect of isoflurane on A23187-mediated relaxation was evident only at higher concentrations of A23187. Differences in our findings and those of Toda et al. [2] may be related to differences in vessel types examined and experimental methods. Since relaxation due to either ACh or A23187 is attenuated by isoflurane, the effect of isoflurane appears to be at or distal to endothelial influx of Ca2+.

Whether volatile anesthetics have an effect distal to the endothelium in the NO-cGMP signaling pathway has been controversial. Earlier studies [1-3] demonstrated no effect of volatile anesthetics on relaxation due to endothelium-independent NO generators (nitroprusside, nitroglycerin). Blaise and To [6], however, suggested that isoflurane interferes with NO-mediated relaxation at a site distal to the endothelium, either by diminishing the stability of NO or its action on VSM, presumably VSM guanylate cyclase. Recent studies on the effect of anesthetics on stimulation of guanylate cyclase itself have differed in concluding that halothane and isoflurane have an attenuating effect at the VSM enzyme level [4,5,17] or the contrary [15,16]. In an attempt to explain the differences in findings, Jing et al. [17] suggested that because the volatile anesthetics have a weak interaction with VSM guanylate cyclase, enough equilibration time must be provided between anesthetics and tissue samples prior to exposure to an agonist of the enzyme, in order to demonstrate attenuation of the enzyme activity. Short equilibration time such as in studies by Johns et al. [15] and Zuo et al. [16] may explain lack of demonstration of attenuation of soluble guanylate cyclase activity by volatile anesthetics in their studies.

In our model, each vessel was equilibrated with a volatile anesthetic for more than enough time required to achieve equilibration before the vessel was exposed to a vasodilator. Using this approach, isoflurane appears to act at two different steps distal to the endothelial increase of Ca2+. First, the differential sensitivities of SNP-induced vasodilation versus A23187-induced vasodilation to the attenuating effect of isoflurane suggest that isoflurane may interfere at an intermediate step between endothelial increase in Ca2+ and guanylate cyclase. This step may involve NO production or transport. NO production may be impaired by inhibiting endothelial influx of Ca2+, by a direct action on endothelial NO synthase, by interfering with the interaction of NO synthase with its cofactors (calmodulin, reduced nicotinamide adenine dinucleotide phosphate, and Ca2+) and/or its substrates (L-arginine and oxygen). NO transit to the VSM may be impaired by enhancing NO breakdown and/or, if thiols are essential in NO transit [20], by interfering with the interaction of NO and a thiol. While our data do not distinguish between these possibilities, the study by Blaise and To [6] appears to suggest an action other than that on NO production. They separated NO production and the distal steps by using endothelial cell-covered beads as donors and endothelium-denuded rings as detectors of NO and demonstrated that isoflurane did not affect NO production, but only worked distally to it. Yoshida and Okabe [21] were the first to demonstrate production of oxygen-derived free radicals by a volatile anesthetic, namely sevoflurane. Recently, we demonstrated that in rabbit coronary microvessels, isoflurane-mediated contraction of coronary microvessels is abolished by scavengers of oxygen-derived free radicals [22]. Oxygen-derived free radicals can inactivate NO [23]. Conceivably, isoflurane may inactivate NO via production of oxygen-derived free radicals in rat coronary microvessels.

Second, the fact that isoflurane 2% attenuates SNP-mediated vasodilation, but not Br-cGMP-mediated vasodilation suggests that a high concentration of isoflurane (2%) may also attenuate the activity of the VSM guanylate cyclase. An alternative explanation may be that isoflurane interferes with reduction of SNP to release NO; and this possibility is not completely ruled out by our findings. Because reduction of nitroprusside to NO is enhanced by reducing agents [10], oxygen-derived free radicals could interfere with the reduction process. Another explanation may be that isoflurane enhances phosphodiesterase and cGMP breakdown. However, we have previously shown that isoflurane 2% attenuates vasodilation by the phosphodiesterase inhibitor RO 20-1724 [14].

The VSM-soluble guanylate cyclase is a sulfhydryl enzyme with a heme moiety [24]. Its activity is modulated by the ratio of reduced and oxidized SH groups [24] and inhibited by a metalloporphyrin, usually ferroprotoporphyrin IX or heme [25]. No interacts with the iron of heme, removing the iron from the plane of the porphyrin ring, and the demetallation activates guanylate cyclase [26,27]. Additionally, hydroxyl radical [27] and prostaglandin endoperoxides [28] may interact with the regulatory thiol groups of the enzyme and increase its activity. However, prolonged exposure of the enzyme to the oxidants results in the loss of its activity [24]. Conceivably, isoflurane may attenuate the activity of guanylate cyclase by interfering with the interaction of NO with the heme moiety of the enzyme and/or altering the oxidation status of the regulatory thiol groups of the enzyme.

Studies using conductance vessels demonstrating an effect of volatile anesthetics at the guanylate cyclase level tended to show that halothane has a stronger attenuating effect than isoflurane [5,17]. However, in our preparation of resistance coronary arteries, we could only demonstrate a modest effect of isoflurane at the guanylate cyclase level, but no effect of halothane. It may be postulated that there are different forms of guanylate cyclase in conductance and resistance vessels, accounting for different response profiles to volatile anesthetics.

Finally, we have demonstrated that the attenuating effects of isoflurane and halothane are reversible and nonlingering once the anesthetics are gone. Any proposed mechanism of a volatile anesthetic-mediated attenuation of endothelium-dependent vasodilation will have to be consistent with the transient nature of the effect.

Any suggestion of clinical implications of our and others' findings of volatile anesthetic-mediated attenuation of endothelium-dependent dilation must be tempered by the in vitro nature of the studies performed thus far. Given this proviso, one may postulate how volatile anesthetic may affect exogenous vasodilators that use the NO-cGMP pathway or endogenous mechanisms that generate NO to elicit vasodilation. Among vasodilators in common clinical use, none are endothelium-dependent. Nitroglycerin and SNP are endothelium-independent donors of NO. While a high concentration of isoflurane may attenuate nitroglycerin- or SNP-mediated dilation of resistance coronary arteries, the modest effect of isoflurane may be easily overcome by simply increasing the dose of either vasodilator and may not be clinically appreciable.

Flow-mediated vasodilation is an example of an endogenous NO-generating mechanism [8]. It is one of the important determinants of coronary flow distribution, along with metabolic, myogenic, and neurohumoral mechanisms [29]. Halothane, which affects only the receptor-mediated endothelium-dependent vasodilation in resistance coronary arteries, may not have any effect on flow-mediated NO synthesis, which by-passes the endothelial agonist receptor. On the other hand, isoflurane, which may have an effect distal to endothelial synthesis of NO, may have an attenuating effect on flow-mediated vasodilation. The implications of our findings on actual coronary blood flow distribution would have to be considered in conjunction with anesthetic effects on other determinants of flow distribution, such as metabolic, myogenic, and neurohumoral mechanisms.

In conclusion, we have demonstrated, in rat coronary microvessels, that halothane and isoflurane attenuate endothelium-dependent vasodilation. Whereas halothane has a mild effect at the endothelial receptor level, isoflurane impairs at least two distinct steps in the NO-cGMP pathway. The first of these is at or distal to the endothelial increase in Ca2+ that activates NO synthase and proximal to VSM guanylate cyclase. The second may involve weak inhibition of guanylate cyclase. These two steps appear to have different sensitivities to the effect of isoflurane.

The authors would like to express their gratitude to Hang Lee, PhD, of Harvard Medical School, for assistance with statistical analysis; Leo Hannenberg for assistance with computer software; and John Zeind, MS, for assistance with gas chromatography.

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